Mycoplasma pneumoniae is a significant human respiratory tract pathogen, causing bronchitis and atypical or walking pneumonia. M. pneumoniae accounts for 20% of all community-acquired pneumonia and is the leading cause of pneumonia in older children and young adults. Serologic testing is the primary method for diagnosis due to the significant challenges posed by direct culture but suffers from severe limitations, including the need for paired sera obtained at separate physician visits, and is thus impractical for rapid diagnosis. PCR can exhibit high sensitivity and yield positive detection sooner but is prone to false-negatives from reaction inhibitors. The inability to provide rapid and definitive diagnosis delays initiation of appropriate treatment, prolongs morbidity, and increases the likelihood of continued transmission, secondary infections, and long-term sequelae, including chronic lung disease associated with COPD and asthma. Lack of a simple, reliable, rapid diagnostic test is thus a critical barrier to improved control of M. pneumoniae disease. Application of nanotechnology to biosensor development is yielding direct, rapid, and sensitive pattern-recognition approaches for detection of infectious agents. We have shown that nanofabrication by glancing angle vapor deposition produces Ag nanorod arrays (NA) exhibiting extremely high electromagnetic field enhancements for surface-enhanced Raman spectroscopy (SERS). Paired with chemometric analysis this platform can rapidly detect and distinguish with outstanding sensitivity and specificity the Raman spectra of viruses and mycoplasmas, and shows great promise in its potential to improve diagnosis of M. pneumoniae infections. Our overall goal is to advance the application of NA-SERS to rapid and sensitive mycoplasma detection in clinical specimens, building upon critical published data that demonstrate its capacity to detect M. pneumoniae at clinically relevant sensitivity in simulated and true clinical throat swab samples. Our application has two specific aims, shifting the project from proof-of-concept toward clinical application: (1) expand analysis of spiked and control samples using throat swabs from a broader cohort, including direct comparison to qPCR; analyze spectral signatures of commensal mycoplasmas and assess their impact on M. pneumoniae detection; and assess NA- SERS discriminatory ability in mixed infections with other bacterial pathogens including Chlamydophila pneumoniae; and (2) test by NA-SERS, true clinical throat swab samples previously established as positive or negative for M. pneumoniae by PCR and culture, in unblinded and blinded analyses; and characterize M. pneumoniae isolates obtained globally to determine whether strain diversity impacts detection by NA-SERS in spiked throat swab samples. PUBLIC HEALTH RELEVANCE: Mycoplasma pneumoniae is a significant human respiratory tract pathogen, accounting for 20- 30% of all community-acquired pneumonia and the leading cause of pneumonia in older children and young adults. The inability to provide rapid and definitive diagnosis delays initiation of appropriate treatment, prolongs morbidity, and increases the likelihood of continued transmission and long-term complications, including COPD and asthma. Lack of a simple and reliable rapid diagnostic test is thus a critical barrier to improved control of M. pneumoniae disease, and our goal is to advance the development of a nanotechnology-based platform for sensitive and expedient detection of M. pneumoniae in clinical specimens.